Lupus Information and Courses from MediaLab, Inc.

Autoimmune diseases are a group of disorders where the body's immune system malfunctions and attacks its own tissues. One aspect of these diseases is the formation of antibodies that are directed to self-antigens (autoantibodies). Autoimmune diseases can be divided into two general groups: Organ specific, where the autoantibodies attack a specific organ, and Non-organ specific (or systemic), where the autoantibodies attack multiple organ systems. An example of an organ specific autoimmune disease is Hashimoto thyroiditis where autoantibodies damage the thyroid gland. An example of a systemic autoimmune disease is systemic lupus erythematosus (SLE) where the autoantibodies may attack any organ in the body.

Systemic autoimmune rheumatic diseases (SARDs) are a group of autoimmune diseases that include:Rheumatoid arthritis (RA) Systemic lupus erythematosus (SLE) (and subsets of lupus) Sjögren syndrome (SjS) Systemic sclerosis (SSc) Polymyositis (PM) Dermatomyositis (DM)

Pattern observed by indirect immuno fluorescence Antigen Disease(s) in which antibodies are seen Routine tests used to confirm specific antibody Homogeneous Double stranded DNA (dsDNA) Characteristic of SLE, lower levels in other rheumatic diseases IFA or CZ using Crithidia luciliae, RIA, ELISA, Addressable Laser Bead Assay (ALBIA) Nucleosome or Chromatin SLE, Drug-induced LE ELISA Histone Drug-induce LE, SLE ELISA, ALBIA Unusual Homogeneous Nuclear Membrane Lupoid hepatitis ELISA for gp-210 Speckled Sm (Smith) Marker antibody for SLE Immunodiffusion (ID), ELISA, ALBIA U1-RNP High levels in MCTD and SLE, low levels in other rheumatic diseases ID, ELISA, ALBIA Speckled (and/or SSA pattern if using HEp-2000®) Can also be ANA negative SS-A/Ro High prevalence in Sjögren syndrome sicca complex, lower prevalence in other rheumatic diseases With HEp-2000 characteristic ANA pattern is confirmatory, others confirm with ID, ELISA, ALBIA Fine speckled or ANA negative Ro52 Sjögren syndrome, myositis, Neonatal Lupus ELISA, ALBIA Fine speckled (sometimes with nucleolar staining as well) SS-B/La High prevalence in Sjögren syndrome sicca complex, lower prevalence in other rheumatic diseases ID, ELISA, ALBIA Fine speckled, Homogeneous, Nucleolar Scl-70 Marker antibody for Scleroderma ID, ELISA, ALBIA Cell Cycle Dependent Speckled PCNA Marker antibody for SLE ID, ELISA, ALBIA Coarse Speckled Nuclear Matrix Seen in some patients with evolving connective tissue disease NONE 3-20 dots NSp I, sp-100, MND, PBC 95 Associated with Primary Biliary Cirrhosis ELISA, ALBIA Cell Cycle Dependent Speckled with speckling in metaphase mitotics NSp II, CENP F Some association with malignancies NONE Staining in cleavage furrow between dividing cells Midbody Unknown Confirm by staining pattern Centromere CENP A, CENP B, CENP C Seen in 57-82% of patients with limited form (CREST) of scleroderma and Raynaud phenomenon Confirmed by staining pattern ELISA, ALBIA Nucleolar Fibrillarin (Clumpy nucleolar) Scleroderma ELISA, ALBIA RNA polymerase I, NOR-90, others? (Speckled nucleolar) Scleroderma and other connective tissue diseases ELISA, ALBIA PM-1 (PM/Scl), others?(Smooth nucleolar) Polymyositis/Scleroderma overlap ELISA, ALBIA

American College of Rheumatology, Committee on Rheumatologic Care, Position Statement, Methodology of Testing for Antinuclear Antibodies; Feb, 2009. Available at http://www.rheumatology.org/search/search.asp accessed on June 16, 2010Anuradah V, Chopra A, Sturgess A, Edmonds J. Cost-effective screening method for antinuclear antibody detection. Asian Pacific League of Associations for Rheumatology. 2004(7):13-18.Arbuckle MR, McClain MT, Rubertone MV, et al. Development of autoantibodies before the clinical onset of systemic lupus erythematosus. N Engl J Med. Oct 16 2003;349(16):1526-1533.Bossuyt X, Frans J, Hendrickx A, Godefridis G, Westhovens R, Marien G. Detection of Anti-SSA Antibodies by Indirect Immunofluorescence. Clin Chem. 10 7 2004;50(12):2361-2369.Clinical and Laboratory Standards Institute (formerly NCCLS); Quality Assurance of Laboratory Tests for Autoantibodies to Nuclear Antigens: (1) Indirect Fluorescence Assay for Microscopy and (2) Microtiter Enzyme Immunoassay Methods; Approved Guidelines - Second Edition. CLSI I/LA2-A2. 2006;26(13).Fritzler MJ, Hanson C, Miller J, Eystathioy T. Specificity of autoantibodies to SS-A/Ro on a transfected and overexpressed human 60 kDa Ro autoantigen substrate. J.Clin.Lab.Anal. 2002;16:103-108.Fritzler MJ, Miller BJ. Detection of autoantibodies to SS-A/Ro by indirect immunofluorescence using a transfected and overexpressed human 60 kD Ro autoantigen in HEp-2 cells. J.Clin.Lab.Anal. 1995;9:218-224.Fritzler MJ, Wall W, Gohill J, Kinsella TD, Humbel RL. The Detection of Autoantibodies on HEp-2 Cells Using an Indirect Immunoperoxidase Kit (Colorzyme®). Diag Immunol. 1986;4:217-221. Keech CL, Howarth S, Coates T, Rischmueller M, McCluskey J, Gordon TP. Rapid and sensitive detection of anti-Ro (SS-A) antibodies by indirect immunofluorescence of 60kDa Ro HEp-2 transfectants. Pathology. 1996;28:54-57.Keech CL, McCluskey J, Gordon TP. Transfection and overexpression of the human 60-kDa Ro/SS-A autoantigen in HEp-2 cells. Clin.Immunol.Immunopathol. 1994;73:146-151.Kroshinsky D, Stone JH, Bloch DB, Sepehr A. Case records of the Massachusetts General Hospital. Case 5-2009. A 47-year-old woman with a rash and numbness and pain in the legs. N Engl J Med. Feb 12 2009;360(7):711-720. McCarty, G.A., Valencia, D.W., and Fritzler, M.J., Antinuclear Antibodies-Contempory Techniques and Clinical Application to Connective Tissue Disease. New York: Oxford University Press, Inc. 1984. Murray DL, Homburger HA, Horvat RT, Snyder MR, College of American Pathologists; S-C 2009: Antinuclear Antibody Screening Methods; CAP Surveys S-C Diagnostic Immunology;2009 Pollock W, Toh BH. Routine immunofluorescence detection of Ro/SS-A autoantibody using HEp-2 cells transfected with human 60 kDa Ro/SS-A. J.Clin.Pathol. 1999;52:684-687.Singer, M. and Berg, P., Genes & Genomes-A Changing Perspective. Mill Valley, CA: University Science Books. 1991.Sullivan KE. The complex Genetic Basis of Systemic Lupus Erythematosus, Reprint from 1999 and 2000; Lupus Foundation, Available at http://www.lupus.org/education/articles/geneticbasis.html Accessed June 16, 2010.Wallace DJ. New methods for antinuclear antibody testing: does it cut costs and corners without jeopardizing clinical reliability? Nat Clin Pract Rheumatol. Aug 2006;2(8):410-411.Willcocks LC, Carr EJ, Niederer HA, et al. A defunctioning polymorphism in FCGR2B is associated with protection against malaria but susceptibility to systemic lupus erythematosus. Proc Natl Acad Sci U S A. Apr 27 2010;107(17):7881-7885.

The presence of an increased amount of protein in a urine specimen is often the first indicator of renal disease. Proteinuria may signal severe kidney damage, be a warning of impending kidney involvement, or be transient and unrelated to the renal system. Further quantitative testing of urine for protein may be needed to determine the significance of the proteinuria. Proteinuria related to kidney impairment may be due to glomerular membrane damage caused by toxic agents, immune complexes found in lupus erythematosus, or streptococcal glomerulonephritis. The amount of protein present in urine samples from patients with glomerular damage usually ranges from 10-40 mg/dL. If the urinary protein is due to a disorder that affects tubular reabsorption, the urine protein quantities will be much greater. In patients with multiple myeloma, proteinuria is due to the excretion of the Bence Jones protein. This low molecular weight protein produced by a malignant clone of plasma cells circulates in the blood and is filtered in the kidneys in quantities exceeding the tubular capacity. This excess protein is excreted in the urine.

Proteinuria related to kidney impairment may be due to glomerular membrane damage caused by toxic agents, immune complexes found in lupus erythematosus, or streptococcal glomerulonephritis. The amount of protein present in urine samples from patients with glomerular damage usually ranges from 10-40 mg/dl. If the urinary protein is due to a disorder that affects tubular reabsorption, the urine protein quantities will be much greater. In patients with multiple myeloma, proteinuria is due to the excretion of the Bence Jones protein. This low molecular weight protein produced by a malignant clone of plasma cells circulates in the blood and is filtered in the kidneys in quantities exceeding the tubular capacity. This excess protein is excreted in the urine.

Severe proteinuria (greater than 3.5 g/day) is characteristically seen in patients with glomerulonephritis, lupus nephritis, lipoid nephrosis, and severe venous congestion of the kidney. Moderate proteinuria (0.5-3.5g/day) is seen in nephrosclerosis, multiple myeloma, diabetes nephropathy, malignant hypertension, and pyelonephritis with hypertension. Mild proteinuria (less than 0.5 g/day) may be seen with polycystic kidneys, chronic pyelonephritis, benign orthostatic proteinuria, and some renal tubular diseases. Transient proteinuria can also be due to physiologic conditions such as stress, exercise, cold exposure, and fever, in the absence of renal disease.

PT and/or aPTT may be prolonged for a number of reasons. Prolonged PT causes include: Warfarin therapy Liver disease Disseminated intravascular coagulation (DIC) Vitamin K deficiency Liver conditions such as cirrhosis or hepatitis Inadequate level of Factors I, II, V, VII, and/or XProlonged aPTT causes include: Presence of heparin Liver disease, other liver conditions Vitamin K deficiency Hemophilias DIC von Willebrand disease Lupus anticoagulant Inadequate levels of Factors I, II, V, VIII, IX, X, XI, and/or XII

No single screening test can detect all lupus anticoagulant-positive (LA-positive) patients. Several tests are available and at least two should be employed to verify the presence of LA. Before any LA screening test is done, a thrombin time (TT) should be performed to rule out therapeutic heparin or the presence of a thrombin (factor-II) inhibitor.These are some of the LA screening procedures that can then be used: Dilute Russell's Viper Venom time (DRVVT). This test utilizes a reagent containing venom from the viper Vipera russelli (which activate factor V and X), low levels of phospholipids, and calcium ions in a clotting time test. The DRVVT test principle is based on the idea that the reagents can help to identify the antibody's dependence on phospholipids . Platelet neutralization procedure. This assay will show the dependence on phospholipids for the lupus anticoagulant to take effect. This can be performed using the aPTT based technique, with the DRVVT test, or using Taipan snake venom time tests. Kaolin clotting time or silica clotting time Hexagonal Phospholipid test (HPP). This is a similar assay to the platelet neutralization procedure, but thought to be more sensitive.

For the diagnosis of lupus anticoagulant, the International Society on Thrombosis and Hemostasis has set a protocol of diagnostic criteria that should be met. This includes the following requirements: The patient sample must show abnormal phospholipid-dependent reactions in the coagulation lab. The patient sample must show inhibition of clotting after the mixing study test has been performed. The patient sample must be proven to have an inhibitor and not a factor deficiency. The patient must have a definitive phospholipid-dependent antibody and not a specific factor inhibitor.

As the name implies, coagulation inhibitors (also called circulating anticoagulants) interfere with normal blood coagulation. Coagulation inhibitors may be congenital or acquired (developing in patients during the course of a disease) and are almost always immunoglobulins, either IgG or IgM. There are two types of inhibitors: those directed toward a coagulation factor (or multiple factors) and the lupus anticoagulant. Lupus anticoagulant is one of the more commonly encountered coagulation inhibitors. It is also known as antiphospholipid antibody because it is directed toward phospholipids. Lupus anticoagulant is usually an IgG antibody. It differs from factor-specific inhibitors in that lupus anticoagulant causes thrombosis and abnormal clotting while factor-specific inhibitors cause serious bleeding.

1. Aniara Learning Center. Coagulation Corner. Mixing Studies: To correct or not correct-that is the question. June 2009. http://www.aniara.com/learning-center/Coagulation-Corner/articles/2009/01/mixing-studiesto-correct-or-not-correct.aspx.2. Bethel, M and Adcock, D: Laboratory evaluation of a prolonged APTT and PT. Lab Med, 285, May 2004.3. Devreese KM. Interpretation of normal plasma mixing studies in the laboratory diagnosis of lupus anticoagulants. Thromb Res 2007;119:369-76.4. Harmening, D. Clinical Hematology and Fundamentals of Hemostasis. 5th edition. F.A. Davis, 2009.5. Katrien M.J. Devreese, Interpretation of normal plasma mixing studiesin the laboratory diagnosis of lupus anticoagulants, ThrombosisResearch, Volume 119, Issue 3, 2007, Pages 369-376, ISSN 0049-3848,DOI: 10.1016/j.thromres.2006.03.012.(http://www.sciencedirect.com/science/article/B6T1C-4JYKP68-1/2/12550b597f6b88b11e09b26e74963d4f)Keywords: Lupus anticoagulants; Mixing tests; Percent correction formula; Rosner index6. McKenzie, S. Clinical Laboratory Hematology. 2nd edition. Pearson, 2010.7. National Committee for Clinical Laboratory Standards. Determination of Factor Coagulant Activities, H48A. NCCLS, 1997.8. Santora SA, Eby CS, Chapter 106: Laboratory evaluation of hemostatic disorders. Pages 1841-1844. In: Hoffman R, Benz, EJ, Jr et. al Hematology. Basic Prinicples and Practice. 3rd edition. Churchill Livingstone. 2000.9. Vancott, E and Laposata, M: Coagulation, Fibrinolysis and Hypercoagulation. 2001.

The traditional CRP test uses immunoassay methods that are sensitive to concentrations of 5-20 mg/L. The hs-CRP test, with its increased sensitivity, is able to detect C-reactive protein in lower levels, 0.5-10.0 mg/L. As with most risk markers, the results of hs-CRP testing are generally interpreted on a relative scale; the higher the value, the higher the risk of a future cardiovascular event.The American Heart Association and Centers for Disease Control and Prevention has defined risk groups with hs-CRP as follows: Low risk: < 1.0 mg/L Average risk: 1.0 to 3.0 mg/L High risk: > 3.0 mg/L It is important to note that hs-CRP assays are measuring the same protein as traditional CRP assays. Thus, in patients with active inflammation (such as chronic, active arthritis; lupus; infection; etc.) hs-CRP values would be expected to be high and would not necessarily implicate cardiovascular risk. If values greater than 10 mg/L are seen in repeated measurements, a non-cardiovascular cause should be considered. Taking anti-inflammatory drugs (NSAIDs, aspirin, etc.) or the statin-class of cholesterol-lowering drugs may reduce CRP levels in patients. This is not an artifact, but is thought to be an effect of treating the underlying inflammatory process.

Illustrated in the image is a phagocyte devouring several erythrocytes. This uncommon phenomenon occurs in the bone marrow and in the spleen as part of the process of erythrocyte destruction. Erythrophagocytosis is found in histological sections of the spleen in cases of hemolytic anemia. This phenomenon appears also in splenic sections in lupus erythematosis, and in rheumatoid arthritis. Our example is from a patient with a myeloproliferative disorder and is a rare example of a circulating erythrophagocytic cell in the peripheral blood.